In sample analysis instrumentation, and especially in separation systems such as liquid chromatography and capillary electrophoresis systems, smaller dimensions will generally result in improved performance characteristics and at the same time result in reduced production and analysis costs. In this regard, miniaturized separation systems provide more effective system design, result in lower overhead due to decreased instrumentation sizing and additionally enable increased speed of analysis, decreased sample and solvent consumption and the possibility of increased detection efficiency.
Accordingly, several approaches towards miniaturization for liquid phase analysis have developed in the art; the conventional approach using drawn fused-silica capillary, and an evolving approach using silicon micromachining. What is currently thought of as conventional in miniaturization technology is generally any step toward reduction in size of the analysis system.
In conventional miniaturized technology the instrumentation has not been reduced in size; rather, it is the separation compartment size which has been significantly reduced. As an example, micro-column liquid chromatography (.mu.LC) has been described wherein columns with diameters of 100-200 .mu.m are employed as compared to prior column diameters of around 4.6 mm.
Another approach towards miniaturization has been the use of capillary electrophoresis (CE) which entails a separation technique carried out in capillaries 25-100 .mu.m in diameter. CE has been demonstrated to be useful as a method for the separation of small solutes. J. Chromatogr. 218:209 (1981); Analytical Chemistry 53:1298 (1981). In contrast, polyacrylamide gel electrophoresis was originally carried out in tubes 1 mm in diameter. Both of the above described "conventional" miniaturization technologies (.mu.LC and CE) represent a first significant step toward reducing the size of the chemical portion of a liquid phase analytical system. However, even though experimentation with such conventional miniaturized devices has helped to verify the advantages of miniaturization in principal, there nevertheless remain several major problems inherent in those technologies.
For example, there remains substantial detection limitations in conventional capillary electrophoresis technology. For example, in CE, optical detection is generally performed on-column by a single-pass detection technique wherein electromagnetic energy is passed through the sample, the light beam travelling normal to the capillary axis and crossing the capillary only a single time. Accordingly, in conventional CE systems, the detection path length is inherently limited by the diameter of the capillary.
Given Beer's law, which relates absorbance to the path length through the following relationship: EQU A=.epsilon.*b*C
where:
A=the absorbance PA1 .epsilon.=the molar absorptivity, (1/m*cm) PA1 b=path length (cm) PA1 C=concentration (m/1)
it can be readily understood that the absorbance (A) of a sample in a 25 .mu.m capillary would be a factor of 400.times. less than it would be in a conventional 1 cm path length cell as typically used in UV/V is spectroscopy.
In light of this significant detection limitation, there have been a number of attempts employed in the prior art to extend detection path lengths, and hence the sensitivity of the analysis in CE systems. In U.S. Pat. No. 5,061,361 to Gordon, there has been described an approach entailing micro-manipulation of the capillary flow-cell to form a bubble at the point of detection. In U.S. Pat. No. 5,141,548 to Chervet, the use of the Z-shaped configuration in the capillary, with detection performed across the extended portion of the Z has been described. Yet another approach has sought to increase the detection path length by detecting along the major axis of the capillary (axial-beam detection). Xi et al., Analytical Chemistry 62:1580 (1990).
In U.S. Pat. No. 5,273,633 to Wang, a further approach to increased detection path lengths in CE has been described where a reflecting surface exterior of the capillary is provided, the subject system further including an incident window and an exit window downstream of the incident window. Under Wang, light entering the incident window passes through a section of the capillary by multiple internal reflections before passing through the exit window where it is detected, the subject multiple internal reflections yielding an effective increase in path length. While each of the aforementioned approaches has addressed the issue of extending the path length, each approach is limited in that it entails engineering the capillary after-the-fact or otherwise increasing the cost of the analysis.
A second major drawback in the current approach to miniaturization involves the chemical activity and chemical instability of silicon dioxide (SiO.sub.2) substrates, such as silica, quartz or glass, which are commonly used in both CE and .mu.LC systems. More particularly, silicon dioxide substrates are characterized as high energy surfaces and strongly adsorb many compounds, most notably bases. The use of silicon dioxide materials in separation systems is further restricted due to the chemical instability of those substrates, as the dissolution of SiO.sub.2 materials increases in basic conditions (at pHs greater than 7.0).
To avoid the problems arising from the inherent chemical activity of silicon dioxide materials, prior separation systems have attempted chemical modifications to the inner silica surface of capillary walls. In general, such post-formation modifications are difficult as they require the provision of an interfacial layer to bond a desired surface treatment to the capillary surface, using, for example, silylating agents to create Si--O--Si--C bonds. Although such modifications may decrease the irreversible adsorption of solute molecules by the capillary surfaces, these systems still suffer from the chemical instability of Si--O--Si bonds at pHs above 7.0. Accordingly, chemical instability in SiO.sub.2 materials remains a major problem.
However, despite the recognized shortcomings with the chemistry of SiO.sub.2 substrates, those materials are still used in separation systems due to their desirable optical properties. In this regard, potential substitute materials which exhibit superior chemical properties compared to silicon dioxide materials are generally limited in that they are also highly adsorbing in the UV region, where detection is important.
In order to avoid some of the substantial limitations present in conventional .mu.LC and CE techniques, and in order to enable even greater reduction in separation system sizes, there has been a trend towards providing planarized systems having capillary separation microstructures. In this regard, production of miniaturized separation systems involving fabrication of microstructures in silicon by micromachining or microlithographic techniques has been described. See, e.g. Fan et al., Anal. Chem. 66(1):177-184 (1994); Manz et al., Adv. Chrom. 33:1-66 (1993); Harrison et al., Sens. Actuators, B10 (2): 107-116 (1993); Manz et al., Trends Anal. Chem. 10 (5); 144-149 (1991); and Manz et al., Sensors and Actuators B (Chemical) B1 (1-6): 259-255 (1990).
State-of-the-art chemical analysis systems for use in chemical production, environmental analysis, medical diagnostics and basic laboratory analysis must be capable of complete automation. Such a total analysis system (TAS) (Fillipini et al (1991) J. Biotechnol. 18:153; Garn et al (1989) Biotechnol. Bioeng. 34:423; Tshulena (1988) Phys. Ser. T23:293; Edmonds (1985) Trends Anal. Chem. 4:220; Stinshoff et al. (1985) Anal. Chem. 57:114R; Guibault (1983) Anal. Chem Symp. Ser. 17:637; Widmer (1983) Trends Anal. Chem. 2:8) automatically performs functions ranging from introduction of sample into the system, transport of the sample through the system, sample preparation, separation, purification and detection, including data acquisition and evaluation. Miniaturized total analysis systems have been referred to as ".mu.-TAS."
Recently, sample preparation technologies have been successfully reduced to miniaturized formats. Gas chromatography (Widner et al. (1984) Int. J. Environ. Anal. Chem. 18:1), high pressure liquid chromatography (Muller et al. (1991) J. High Resolut. Chromatogr. 14:174; Manz et al., (1990) Sensors & Actuators B1:249; Novotny et al., eds. (1985) Microcolumn Separations: Columns, Instrumentation and Ancillary Techniques (J. Chromatogr. Library, Vol. 30); Kucera, ed. (1984) Micro-Column High Performance Liquid Chromatography, Elsevier, Amsterdam; Scott, ed. (1984) Small Bore Liquid Chromatography Columns: Their Properties and Uses, Wiley, N.Y.; Jorgenson et al., (1983) J. Chromatogr. 255:335; Knox et al. (1979) J. Chromatogr. 186:405; Tsuda et al. (1978) Anal. Chem. 50:632) and capillary electrophoresis (Manz et al. (1992) J. Chromatogr. 593:253; Manz et al. Trends Anal. Chem. 10:144; Olefirowicz et al. (1990) Anal. Chem. 62:1872; Second Int'l Symp. High-Perf. Capillary Electrophoresis (1990) J. Chromatogr. 516; Ghowsi et al. (1990) Anal. Chem. 62:2714) have been reduced to miniaturized formats.
Capillary electrophoresis has been particularly amenable to miniaturization because the separation efficiency is proportional to the applied voltage regardless of the length of the capillary Harrison et al. (1993) Science 261:895-897. A capillary electrophoresis device using electroosmotic fluid pumping and laser fluorescence detection has been prepared on a planar glass microstructure. Effenhauser et al. (1993) Anal. Chem. 65:2637-2642; Burggraf et al. (1994) Sensors and Actuators B20:103-110. In contrast to silicon materials (see, Harrison et al. (1993) Sensors and Actuators B10:107-116), polyimide has a very high breakdown voltage, thereby allowing the use of significantly higher voltages.
The use of micromachining techniques to fabricate separation systems in silicon provides the practical benefit of enabling mass production of such systems. In this regard, a number of established techniques developed by the microelectronics industry involving micromachining of planar materials, such as silicon, exist and provide a useful and well accepted approach to miniaturization. Examples of the use of such micromachining techniques to produce miniaturized separation devices on silicon or borosilicate glass chips can be found in U.S. Pat. No. 5,194,133 to Clark et al.; U.S. Pat. No. 5,132,012 to Miura et al.; in U.S. Pat. No. 4,908,112 to Pace; and in U.S. Pat. No. 4,891,120 to Sethi et al.
Micromachining silicon substrates to form miniaturized separation systems generally involves a combination of film deposition, photolithography, etching and bonding techniques to fabricate a wide array of three dimensional structures. Silicon provides a useful substrate in this regard since it exhibits high strength and hardness characteristics and can be micromachined to provide structures having dimensions in the order of a few micrometers.
Although silicon micromachining has been useful in the fabrication of miniaturized systems on a single surface, there are significant disadvantages to the use of this approach in creating the analysis device portion of a miniaturized separation system.
Initially, silicon micromachining is not amenable to producing a high degree of alignment between two etched or machined pieces. This has a negative impact on the symmetry and shape of a separation channel formed by micromachining, which in turn may impact separation efficiency. Secondly, sealing of micromachined silicon surfaces is generally carried out using adhesives which may be prone to attack by separation conditions imposed by liquid phase analyses. Furthermore, under oxidizing conditions, a silica surface is formed on the silicon chip substrate. In this regard, silicon micromachining is also fundamentally limited by the chemistry of SiO.sub.2. Accordingly, there has remained a need for an improved miniaturized total analysis system which is able to avoid the inherent shortcomings of conventional miniaturization and silicon micromachining techniques.